WO2011025036A1 - 粒子組成物及びこれを有する医薬組成物 - Google Patents
粒子組成物及びこれを有する医薬組成物 Download PDFInfo
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- WO2011025036A1 WO2011025036A1 PCT/JP2010/064816 JP2010064816W WO2011025036A1 WO 2011025036 A1 WO2011025036 A1 WO 2011025036A1 JP 2010064816 W JP2010064816 W JP 2010064816W WO 2011025036 A1 WO2011025036 A1 WO 2011025036A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/38—Albumins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
Definitions
- the present invention relates to a particle composition (for example, polymer micelle) that can be applied as a carrier for a drug delivery system (DDS), and a pharmaceutical composition (for example, polymer micelle) having the particle composition and a drug contained therein. Formulation).
- a particle composition for example, polymer micelle
- DDS drug delivery system
- a pharmaceutical composition for example, polymer micelle
- Patent Documents 1 to 4 disclose DDS in which biopolymers are encapsulated in liposomes composed of lipid bilayers from the viewpoint of enhancing the stability of biopharmaceuticals in vivo.
- the stability of the drug in vivo can be improved by protecting the biopolymer as a drug with a lipid bilayer membrane, but the drug is hardly released from the carrier.
- Such conventional DDS is easily trapped in the reticuloendothelial system such as lung, liver and spleen due to the size of the particle size and the charge of the lipid constituting the lipid bilayer. It may be removed from the blood before reaching.
- the DDS described in Patent Document 4 prevents such trapping in the reticuloendothelial system by making the liposomes stealth, but the drug tends to be hardly released from the carrier.
- DDS using a polymeric micelle composed of a block copolymer unit composed of a hydrophobic polymer chain segment and a hydrophilic polymer chain segment as a carrier significantly reduces the size of DDS particles compared to conventional DDS using liposomes (for example, (Average particle size of 100 nm or less).
- DDS using such a polymeric micelle as a carrier is too difficult to properly transport the drug to the administration target because the action of holding the biopolymer in the DDS particles is too weak. There is a case. In such DDS, the drug may be detached from the carrier during the stock period after production.
- the present invention includes a block copolymer unit having a hydrophobic polymer chain segment and a hydrophilic polymer chain segment, and a plurality of the block copolymer units have the hydrophobic polymer chain segment facing inward and the hydrophilic polymer chain segment.
- a particle composition radially arranged in an outwardly directed state further comprising a charged lipid having a charge opposite to that of the drug to be encapsulated, and by electrostatic binding with the charged lipid While holding the drug in the particle, the charged lipid is arranged in a state of being attracted to the hydrophobic polymer chain segment side, thereby attracting a charged substance having a charge opposite to that of the charged lipid.
- a particle composition in which the outer peripheral surface of the particle is prevented from being charged.
- the present invention provides a pharmaceutical composition comprising the above particle composition and a drug having a charge opposite to that of the charged lipid encapsulated in the particle composition.
- a drug carrier suitable for DDS which has excellent drug retention and prevents the adhesion of biomolecules to the carrier surface that can result in interference with drug delivery to the administration target, and the drug carrier.
- These drug carriers and pharmaceuticals can transport the drug more reliably by the administration object than the conventional DDS.
- FIGS. 1A to 1C are schematic views showing an example of the structure of the particle composition and the pharmaceutical composition of the present invention.
- FIG. 2 is a graph showing the relationship between the absolute value of the zeta potential of the particle composition measured in Test Example 1b and the degree of blood aggregation of the particle composition measured in Test Example 1c.
- FIG. 3 is a graph showing the measurement results of albumin-FITC fluorescence intensity in Test Example 2b.
- FIG. 4 is a diagram showing the results of electrophoresis in Test Example 5a.
- FIG. 5 is a graph for explaining the anticancer activity of the particle composition examined in Test Example 5c.
- FIG. 6 is a graph for explaining the agglutinability in serum of the particle composition examined in Test Example 5d.
- FIG. 7 is a graph showing the relationship between the absolute value of the zeta potential of the particle composition measured in Test Example 5b and the blood aggregation level of the particle composition measured in Test Example 5d.
- FIG. 8 is a graph for explaining organ transferability of a drug examined in Test Example 5f.
- FIG. 1A is a schematic view showing an example of the structure of the particle composition of the present invention (hereinafter sometimes simply referred to as “particle composition”), and FIG. 1B is a partially enlarged view thereof.
- the particle composition 1 contains a block copolymer unit 2 and a charged lipid 3.
- the block copolymer unit 2 has a hydrophilic polymer chain segment 2a and a hydrophobic polymer chain segment 2b.
- the block copolymer unit 2 is a particle with the hydrophobic polymer chain segment 2b facing inward and the hydrophilic polymer chain segment 2a facing outward. Arranged radially in the composition 1.
- the charged lipid 3 has a charge opposite to that of the drug to be encapsulated and is arranged in a state of being drawn toward the hydrophobic polymer chain segment 2b.
- FIG.1 (c) is a schematic diagram which shows an example of the structure of the particulate-form pharmaceutical composition of this invention (henceforth only a "pharmaceutical composition” may be mentioned).
- the pharmaceutical composition 1 ′ has a particle composition 1 and a drug 4 having a charge opposite to that of the charged lipid 3, and the drug 4 electrostatically binds to the charged lipid 3, whereby the drug composition 1 ′ Retained.
- charged lipid refers to an anionic lipid having a negative charge greater than a positive charge in an aqueous medium at physiological pH (eg, pH 7.4), and more than a negative charge in the aqueous medium. It means a cationic lipid having a positive charge. For this reason, in this specification, what is called an amphoteric charged lipid which has a cationic group and an anionic group will also be handled based on said reference
- the charged lipid 3 holds the drug 4 to be encapsulated in the particle composition 1 by electrostatic bonding.
- the charged lipid 3 only needs to have a charge opposite to that of the drug 4 contained in at least the stock environment of the pharmaceutical composition 1 ′ formed from the particle composition 1. Thereby, the drug 4 can be more reliably held in the particle composition 1 during the stock period after manufacture.
- the charged lipid 3 and the drug 4 have opposite charges even in a physiological environment typified by blood (for example, pH 7.4). Thereby, it can prevent more reliably that the chemical
- the charged lipid 3 is arranged in a state of being attracted to the hydrophobic polymer chain segment side 2b by the following mechanism.
- the particle composition 1 is formed by suspending the block copolymer unit 2 and the charged lipid 3 in an aqueous solution. Since the hydrophobic polymer chain segment 2b of the block copolymer unit 2 is hydrophobic, the hydrophobic polymer chain segment 2b does not diffuse in an aqueous solution but is aggregated and present.
- the hydrophilic polymer chain segment 2a can diffuse and move freely in an aqueous solution.
- the block copolymer units 2 are radially arranged in the aqueous solution with the hydrophobic polymer chain segment 2b facing inward and the hydrophilic polymer chain segment 2a facing outward.
- the charged lipid 3 is strongly hydrophobic and has a higher affinity with the hydrophobic polymer chain segment 2b than with the water or hydrophilic polymer chain segment 2a. It will be arranged away from the outer peripheral surface of the object 1. This prevents the outer peripheral surfaces of the particle composition 1 and the pharmaceutical composition 1 ′ from being charged with a charge that can attract a charged substance (for example, a protein in blood) having a charge opposite to that of the charged lipid 3. Is done.
- a charged substance for example, a protein in blood
- the charged lipid 3 and the block copolymer unit 2 are mixed and particle-formed, so that the charged lipid 3 is surrounded by the particle composition 1 as shown in FIG. Rather than being arranged in a continuous state in the direction, they are arranged so as to be interposed between adjacent block copolymer units 2 in the circumferential direction of the particle composition 1.
- the contact between the charged lipids 3 adjacent in the circumferential direction of the particle composition 1 is divided by the block copolymer unit 2.
- a gap G is formed between the charged lipids 3 and the block copolymer unit 2 is disposed in the gap G.
- the binding force between the charged lipid 3 and the block copolymer unit 2 is smaller than the binding force between the charged lipids 3. For this reason, in the particle composition 1, compared with the conventional DDS (liposome) in which the lipid is continuously arranged in the circumferential direction, the particle shape is likely to be collapsed due to the dropping of the charged lipid 3. Further, excessive retention of the drug 4 to be encapsulated in the particles (carrier) is prevented.
- the stealth liposome represented by DDS described in Patent Document 4 is formed by attaching a diblock copolymer to the surface of the main body after forming the main body of the lipid bilayer membrane, it is adjacent in the circumferential direction.
- the contact between the lipids is not interrupted by the block copolymer or the like, but is in a state where the lipids are continuously arranged in the circumferential direction.
- the state in which the outer peripheral surfaces of the particle composition 1 and the pharmaceutical composition 1 ′ are prevented from being charged to attract the chargeable substance is, for example, zeta measured from the particle composition 1 and the pharmaceutical composition 1 ′. It can be specified by the absolute value of the potential being in the range of a predetermined value or less. More specifically, for the pharmaceutical composition 1 ′, the absolute value of the zeta potential is desirably 10 mV or less, for example, 5 mV or less, for example, 3 mV or less, preferably 2 mV or less, more preferably 1 mV or less. It is.
- the absolute value of the zeta potential of the pharmaceutical composition 1 ′ tends to be lower than the absolute value of the zeta potential unique to the particle composition 1 by containing the drug 4 in the particle composition 1. Therefore, for the particle composition 1, the absolute value of the zeta potential is desirably 15 mV or less, for example, 12 mV or less, for example, 6 mV or less, for example, 3 mV or less, preferably 2 mV or less, more preferably 1 mV. It is as follows.
- the zeta potential is adjusted so that the total amount of charged lipid 3 contained in the particle composition 1 or the pharmaceutical composition 1 ′ is 0.1 mg per mL of the buffer solution in a 10 mM HEPES buffer solution having a pH of 7.4. Add and measure.
- the absolute value of the zeta potential is handled as a numerical value obtained by rounding off the decimal point.
- a state where the absolute value of the zeta potential is “2 mV or less” includes a state where the absolute value of the zeta potential is less than 2.5 mV.
- the blood aggregation degree of the particle composition 1 is, for example, 0.2 or less, for example, 0.18 or less, Further, for example, aggregation in blood can be prevented to a level of 0.15 or less, further 0.1 or less, and in some cases 0.05 or less.
- the blood aggregation level of the pharmaceutical composition 1 ′ is, for example, 0.2 or less, for example, 0.16 or less, Aggregation in blood can be prevented to 0.1 or less, and in some cases to 0.05 or less.
- the degree of blood aggregation is calculated as follows. i) In a 10 mM HEPES buffer solution having a pH of 7.4, the composition to be measured (particle composition 1 or pharmaceutical composition 1 ′ so that the total amount of charged lipid 3 is 2.2 mg per mL of the buffer solution) Then, FBS (bovine serum) is further added to 9 mL per 1 mL of the buffer solution to prepare a measurement sample A. ii) A measurement sample B is prepared in the same manner as in i) except that a 10 mM HEPES buffer solution having a pH of 7.4 is used instead of FBS (bovine serum). iii) After allowing the measurement samples A and B to stand at 37 ° C.
- a value obtained by subtracting “absorbance obtained from measurement sample B” from “absorbance obtained from measurement sample A” is defined as a blood agglutination degree.
- the measurement target composition is less likely to aggregate in blood as the value is smaller.
- the ratio of the block copolymer unit 2 to the charged lipid 3 is expressed as a mass ratio, and is desirably 1.0 or more, preferably 1.5 or more, more preferably 2.0 or more, and 50 or less. Desirably, it is preferably 20 or less, more preferably 10 or less.
- the higher the ratio the lower the absolute value of the zeta potential of the particle composition 1 and the pharmaceutical composition 1 '.
- the charged lipid 3 may be any of a simple lipid, a complex lipid, and a derived lipid, and examples thereof include phospholipids, glyceroglycolipids, sphingoglycolipids, sphingoids, and sterols.
- examples of cationic lipids include 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP), N- (2,3-dioleoyloxypropan-1-yl) -N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA), 1, 2-Dimyristyloxypropyl-3-dimethylhydroxyethyl ammonium bromide (DMRIE), 1,2-dioleoyloxypropyl-3-diethylhydroxyethyl ammonium bromide (DORIE), 3 ⁇ - [N— (N′N '-Dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol).
- DOTAP 1,2-dioleoyl-3-trimethylammonioprop
- anionic lipid examples include cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, phosphatidylethylene glycol, and cholesterol succinic acid.
- cardiolipin diacylphosphatidylserine
- diacylphosphatidic acid N-succinylphosphatidylethanolamine (N-succinyl PE)
- phosphatidic acid phosphatidylinositol
- phosphatidylglycerol phosphatidylethylene glycol
- cholesterol succinic acid examples include cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-succinylphosphatidylethanolamine (N-succinyl PE), phosphatidic acid, phosphati
- the hydrophilic polymer chain segment 2a is preferably a segment derived from a water-soluble polymer made of polyethylene glycol or polyoxyethylene.
- the molecular weight of the hydrophilic polymer chain segment 2a is desirably 2,500 Da or more, preferably 5,000 Da or more, more preferably 8,000 Da or more, and desirably 200,000 Da or less. Is 20,000 Da or less, more preferably 15,000 Da or less.
- the hydrophobic polymer chain segment 2b is preferably a segment derived from a polyamino acid chain.
- the number of repeating units of the hydrophobic polymer chain segment 2b is desirably 10 or more, preferably 20 or more, and desirably 200 or less, preferably 100 or less, more preferably 60. Or less.
- the hydrophilic polymer chain segment 2a The size (molecular weight) is preferably larger than the size (number of repeating units) of the hydrophobic polymer chain segment 2b.
- the amino group and You may contain the chargeable substituent represented by the carboxyl group.
- hydrophilic polymer chain segment 2a and the hydrophobic polymer chain segment 2b those obtained by bonding the ends of the main chain through covalent bonds can be used. More specifically, examples of the block copolymer unit 2 include compounds represented by the following general formula (I) and general formula (II).
- the particle composition 1 may be formed of two or more types of block copolymer units 2.
- R 1 and R 3 each independently represent a hydrogen atom or a group represented by R 8 (R 9 ) CH (CH 2 ) q — (provided that , R 8 and R 9 are each independently i) a hydrogen atom, a C 1-6 alkoxy group, an aryloxy group, an aryl C 1-3 oxy group, a cyano group, a carboxyl group, an amino group, C 1-6 An alkoxycarbonyl group, a C 2-7 acylamido group, a tri-C 1-6 alkylsiloxy group, a siloxy group, a silylamino group, ii) taken together or substituted with a C 1-3 alkyl group Forming a substituted ethylenedioxy or propylenedioxy group, or iii) together with the CH group to which they are attached form a formyl group), q is an integer from 0 to 10 and R 2 is hydrogen, saturated or C
- R 7 is a methylene group, n is an integer in the range of 55 to 4,600, x is an integer in the range of 10 to 200, and m is an integer in the range of 0 to 200 (at However, when m is 1 or more, (units of the unit COCHNH) (COR 7 CHNH) is present at random, when m is 2 or more, R 6 each in one block copolymer Are each independently selected at amino acid units are present at random, if R 6 is a hydrogen atom or less 75% of the total R 6), y is 1 or 2, L 1 is -NH- , —O—, —O—Z—NH—, —CO—, —CH 2 —, and —O—Z—S—Z—NH— (wherein Z is independently a C 1 -C 6 alkylene group) L 2 is a linking group selected from —OCO—Z—CO— and —NHCO—Z—CO— (wherein Z is a C 1 to C 6 alkylene group).
- n is preferably an integer of 110 or more, more preferably 180 or more, preferably 460 or less, more preferably 340 or less, and x is preferably 20
- the integer is preferably 100 or less, more preferably 60 or less
- m is preferably an integer of 100 or less, more preferably 60 or less.
- the block copolymer unit 2 is preferably an anionic polymer.
- an anionic polymer as the block copolymer unit 2
- the absolute value of the zeta potential of the particle composition 1 or the pharmaceutical composition 1 ′ is 3 mV or less, and further 2 mV. Below, it becomes easy to control to 1 mV or less.
- an anionic polymer as the block copolymer unit 2
- even if the absolute value of the zeta potential is about the same as that when a neutral polymer is used. , Aggregation in the blood can be prevented more remarkably.
- a polymer having a negative charge larger than a positive charge is anionic, and a polymer having a positive charge larger than a negative charge is cationic, positive.
- a polymer having the same ratio of charge to negative charge is treated as neutral.
- R 5 is —O—
- R 6 is a benzyl group, — (CH 2 ) 4 -phenyl group, or Examples thereof include an unsubstituted or substituted C 4 to C 16 alkyl group with an amino group or a carbonyl group.
- the block copolymer unit 2 can be formed by, for example, purifying a polymer having a hydrophilic polymer chain and a polymer having a polyamino acid chain as they are or, if necessary, to narrow the molecular weight distribution, and then coupling by a known method. .
- N-carboxylic anhydride NCA
- protected amino acids such as ⁇ -benzyl-L-aspartate, ⁇ -benzyl-L-glutamate, N ⁇ -ZL-lysine from its amino terminus it can.
- the particle composition 1 can be formed as follows, for example. First, the block copolymer unit 2, the charged lipid 3, and if necessary, a neutral lipid are dissolved or dispersed in a forming solution containing an organic solvent, and after sufficiently dissolving or dispersing, the organic solvent is removed by evaporation.
- the organic solvent include acetone, dichloromethane, dimethylformamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, and methanol.
- the forming solution can contain two or more organic solvents and may further contain a small amount of water.
- the particle composition 1 can be formed by dispersing / miniaturizing this using means such as ultrasonic irradiation, a high-pressure emulsifier or an extruder.
- a pharmaceutical composition 1 ′ having the particle composition 1 and a drug 4 having a charge opposite to that of the charged lipid 3 encapsulated in the particle composition 1. it can.
- the drug 4 is held in the particle composition 1 by electrostatic coupling with the charged lipid 3.
- the drug 4 can be included in the particle composition by adding it to the forming solution in the process of forming the particle composition 1 or by adding the particle composition 1 to the solution of the drug 4.
- Drug 4 includes an anionic compound having a negative charge greater than a positive charge in an aqueous medium at physiological pH (eg, pH 7.4), and a cationic having a positive charge greater than a negative charge in the aqueous medium.
- the compound is preferably a polymer compound.
- Examples of the polymer compound applicable to the drug 4 include peptides, proteins, sugar chains, and nucleic acids.
- the pharmaceutical composition 1 It is preferable to control the charge ratio between the charged lipid 3 and the drug 4 in a predetermined range.
- the charge ratio is defined by [molar concentration of cationic group in charged lipid 3 contained in particle composition] / [molar concentration of phosphate group in nucleic acid].
- the charge ratio is preferably 0.5 or more, more preferably 1 or more, further preferably 2 or more, and preferably 50 or less, more preferably 20 or less, and even more preferably 10 or less.
- the average particle size of the particle composition 1 and the pharmaceutical composition 1 ′ is preferably 10 nm or more, more preferably 30 nm or more, and preferably 300 nm or less, more preferably 200 nm or less.
- a light scattering particle size measuring device Zetasizer Nano ZS (Malvern Instruments) was used for measurement of the average particle size of the particle composition by dynamic light scattering (DLS).
- Example 1-1 ⁇ -Methoxy- ⁇ -amino-polyethylene glycol having a mass average molecular weight (Mw) of 10000 (hereinafter sometimes referred to as “PEG”) (NOF Corporation) was dissolved in 50 mL of dimethyl sulfoxide, and ⁇ -benzyl- 5.5 g of N-carboxylic acid anhydride (NCA) of L-glutamate (hereinafter sometimes referred to as “PBLG”) (42 equivalents to polyethylene glycol) was added, and the reaction was carried out at 40 ° C. for 24 hours. .
- NCA N-carboxylic acid anhydride
- PBLG L-glutamate
- the reaction solution was dropped into 1 L of a mixed solvent of hexane and ethyl acetate (volume ratio 1: 1) to precipitate a polymer.
- the polymer was collected by vacuum filtration and further dried to obtain 8.6 g of a solid. This was dissolved in 86 mL of DMF, 432 ⁇ L of acetic anhydride was added, and the reaction was performed at 40 ° C. for 24 hours.
- the polymer was precipitated by dropping the reaction solution into 1 L of a mixed solvent of hexane and ethyl acetate (volume ratio 1: 1).
- PEG-PBLG polyethylene glycol-poly ( ⁇ -benzyl-L-glutamate) -Ac block copolymer
- DOTAP cationic charged lipid 1,2-dioleoyl-3-trimethylammoniopropane
- the particle composition 1 is in a state where the hydrophilic polymer chain segment is radially arranged with the outward facing direction, and the charged lipid is attracted to the hydrophobic polymer chain segment side.
- the particle compositions 2 to 6 described later are also in the same state.
- Example 1-2 A particle composition 2 was obtained in the same manner as in Example 1-1 except that phosphatidic acid (hereinafter sometimes referred to as “PA”), which is an anionic charged lipid, was used instead of DOTAP.
- PA phosphatidic acid
- Example 1-3 4 mL of chloroform solution (50 mg / mL) of PEG-PBLG obtained in Example 1-1, 0.5 mL of chloroform solution (40 mg / mL) of DOTAP (Avanti Polar Lipid), which is a cationic charged lipid, and neutral After mixing 0.5 mL of chloroform solution (40 mg / mL) of dioleoylphosphatidylethanolamine (hereinafter sometimes referred to as “DOPE”) (Avanti Polar Lipid), which is a lipid, the solvent is distilled off with a rotary evaporator. And dried under reduced pressure overnight.
- DOPE dioleoylphosphatidylethanolamine
- Example 1-4 A particle composition 4 was obtained in the same manner as in Example 1-3, except that the amount of the PEG-PBLG in chloroform was changed to 2 mL.
- Example 1-5 A particle composition 5 was obtained in the same manner as in Example 1-3, except that the amount of the PEG-PBLG chloroform solution was changed to 1.33 mL.
- Example 1-6 A particle composition 6 was obtained in the same manner as in Example 1-3, except that the amount of the PEG-PBLG in chloroform was changed to 1 mL.
- Example 1-7 By treating the PEG-PBLG obtained in Example 1-1 with an alkali, the benzyl group of the glutamic acid side chain was deprotected to obtain a polyethylene glycol-poly (L-glutamic acid) block copolymer (PEG-pGlu).
- the PEG-pGlu (C8) polymer which is an anionic polymer, is obtained by partially introducing an octyl group (C 8 H 17 ) into the glutamic acid side chain of PEG-pGlu by a condensation reaction using octyl alcohol. It was. From the analysis by 1 H-NMR, the number of octyl groups introduced was 33 per polymer.
- the structural formula of PEG-pGlu (C8) is shown below.
- Example 1-8 A particle composition 8 was obtained in the same manner as in Example 1-7, except that the amount of the PEG-pGlu (C8) methanol solution (50 mg / mL) was changed to 0.5 mL.
- Example 1-9 By treating the PEG-PBLG obtained in Example 1-1 with an alkali, the benzyl group of the glutamic acid side chain was deprotected to obtain a polyethylene glycol-poly (L-glutamic acid) block copolymer (PEG-pGlu).
- the PEG-pGlu (Bn) polymer which is an anionic polymer, was obtained by partially introducing a benzyl group (PhCH 2 ) into the glutamic acid side chain of PEG-pGlu by a condensation reaction using benzyl alcohol. . From the analysis by 1 H-NMR, the number of introduced benzyl groups was 34 per polymer.
- the structural formula of PEG-pGlu (Bn) is shown below.
- Example 1-10 A particle composition 10 was obtained in the same manner as in Example 1-7, except that the amount of the PEG-pGlu (C8) methanol solution (50 mg / mL) was changed to 0.5 mL.
- Comparative Example 1-1 A comparative particle composition C1 was obtained in the same manner as in Example 1-1, except that DOTAP was not added.
- Comparative Example 1-2 A comparative particle composition C2 was obtained in the same manner as in Example 1-3, except that DOTAP was not added.
- Example 1-3 A comparative particle composition C3 was obtained in the same manner as in Example 1-3, except that PEG-PBLG was not added.
- the composition C3 does not contain a block copolymer, but only contains DOTAP, which is a charged lipid, and DOPE, which is a neutral lipid.
- the absolute value of the zeta potential was smaller in the particle compositions 3 to 10 than in the comparative particle composition C3 not containing the block copolymer unit.
- the absolute value of the zeta potential was smaller as the mass ratio of the block copolymer unit to the charged lipid was higher. This result means that the outer peripheral surfaces of the particles of the particle compositions 3 to 10 are in a state in which they are prevented from being charged due to the charged lipid. If the charged lipid content in the particle composition is constant, the outer peripheral surface of the particle becomes less likely to be charged due to the charged lipid as the density of the region constituted by the hydrophilic polymer chain segment increases. Also means.
- the particle composition has low blood aggregation.
- Albumin (pH 7.4) inclusion particle composition [Examples and Test Group 2: Albumin (pH 7.4) inclusion particle composition]
- Albumin-FITC Sigma Aldrich
- bovine-derived albumin was labeled with FITC (fluorescein 5-isothiocyanate) was dissolved in 20 mM HEPES buffer (pH 7.4) to form a 10 mg / mL solution.
- FITC fluorescein 5-isothiocyanate
- Example 2-1 An albumin-containing particle composition was prepared in the same manner as in Example 2, except that the particle composition 2 (containing 40 mg / mL PEG-PBLG and 4 mg / mL PA) was used instead of the particle composition 1. Formed. The average particle size of the particle composition determined by the dynamic light scattering method was 91.6 nm.
- Example 2-2 An albumin-containing particle composition was formed in the same manner as in Example 2 except that the comparative particle composition C1 (containing 40 mg / mL PEG-PBLG) was used instead of the particle composition 1.
- the average particle size of the particle composition determined by the dynamic light scattering method was 118 nm.
- Comparative Example 2-3 Composition of albumin-containing particles in the same manner as in Example 2 except that Comparative Particle Composition C2 (containing 40 mg / mL PEG-PBLG and 2 mg / mL DOPE) was used instead of Particle Composition 1. Formed. The average particle size of the particle composition determined by the dynamic light scattering method was 119 nm.
- Example 2 As shown in Table 4, in Example 2, 94% of albumin was retained in the particles even after centrifugation, but in Comparative Examples 2-1 to 2-3, albumin remained in the particles after centrifugation. I didn't.
- FIG. 3 is a graph showing the measurement results. As shown in FIG. 3, an elution peak was confirmed in the vicinity of fraction 20 in albumin alone and Comparative Example 2-1, whereas an elution peak was confirmed in the vicinity of fraction 10 in Example 2. This means that Example 2 has better retention of albumin in the particles than Comparative Example 2-1.
- Example 3 Dextran-encapsulated particle composition
- Dextran-FITC Sigma Aldrich
- dextran having an average molecular weight of 20,000 was labeled with FITC was dissolved in 20 mM HEPES buffer (pH 7.4) to form a 10 mg / mL solution.
- 20 mM HEPES buffer pH 7.4
- FITC FITC
- 0.2 mL of this dextran solution 0.5 mL of particle composition 1 (containing 40 mg / mL PEG-PBLG and 4 mg / mL DOTAP), and 0.5 mL of 20 mM HEPES buffer (pH 7.4)
- the mixture was allowed to stand at 4 ° C. overnight to form a dextran-encapsulated particle composition.
- the average particle size of the particle composition determined by the dynamic light scattering method was 77.6 nm.
- Dextran is a biopolymer that exhibits anionic properties at pH 7.4.
- a dextran-encapsulated particle composition was prepared in the same manner as in Example 3 except that the particle composition 2 (containing 40 mg / mL PEG-PBLG and 4 mg / mL PA) was used instead of the particle composition 1. Formed.
- the average particle size of the particle composition determined by the dynamic light scattering method was 95.4 nm.
- Example 3-2 A dextran-encapsulated particle composition was formed in the same manner as in Example 3 except that the comparative particle composition C1 (containing 40 mg / mL PEG-PBLG) was used instead of the particle composition 1.
- the average particle size of the particle composition determined by the dynamic light scattering method was 119 nm.
- Comparative Example 3-3 Dextran-encapsulated particle composition in the same manner as in Example 3 except that Comparative Particle Composition C2 (containing 40 mg / mL PEG-PBLG and 4 mg / mL DOPE) was used instead of Particle Composition 1. Formed. The average particle size of the particle composition determined by the dynamic light scattering method was 119 nm.
- Example 3 As shown in Table 5, in Example 3, nearly 40% of dextran was retained in the particles even after centrifugation, but in Comparative Examples 3-1 to 3-3, almost no albumin was present in the particles after centrifugation. It did not remain.
- Example / Test Example Group 4 Albumin (pH 3.3) -encapsulated particle composition
- Albumin from bovine, Sigma Aldrich
- 50 mM glycine buffer pH 3
- 2 mL of this albumin solution, 0.5 mL of particle composition 2 (containing 40 mg / mL PEG-PBLG and 4 mg / mL PA), and 0.5 mL of 20 mM HEPES buffer pH 7.4
- the average particle size of the particle composition determined by the dynamic light scattering method was 78.8 nm. Note that albumin is cationic at pH 3.3.
- Example 4-1 An albumin-containing particle composition was prepared in the same manner as in Example 4 except that the particle composition 1 (containing 40 mg / mL PEG-PBLG and 4 mg / mL DOTAP) was used instead of the particle composition 2. Formed. The average particle size of the particle composition determined by the dynamic light scattering method was 95.1 nm.
- Example 4-2 An albumin-encapsulated particle composition was formed in the same manner as in Example 4 except that the comparative particle composition C1 (containing 40 mg / mL PEG-PBLG) was used instead of the particle composition 2.
- the average particle size of the particle composition determined by the dynamic light scattering method was 115 nm.
- Comparative Example 4-3 Composition of albumin-containing particles in the same manner as in Example 4 except that Comparative Particle Composition C2 (containing 40 mg / mL PEG-PBLG and 1 mg / mL DOPE) was used instead of Particle Composition 2. Formed. The average particle size of the particle composition determined by the dynamic light scattering method was 120 nm.
- Example 4 As shown in Table 6, in Example 4, more than 94% of albumin was retained in the particles after centrifugation, but in Comparative Examples 4-1 to 4-3, the retention rate of albumin after centrifugation was not as high as that of Example 4.
- the encapsulation treatment was performed as follows.
- siRNA was dissolved in 10 mM HEPES buffer (pH 7.4) to prepare a 20 ⁇ M siRNA solution.
- 250 ⁇ L 250 ⁇ L of the particle composition whose concentration was adjusted to satisfy the target charge ratio (+/ ⁇ ) was added, and after mixing, left at 4 ° C. for 2 hours, siRNA encapsulation was performed.
- the “charge ratio (+/ ⁇ )” is [concentration of cationic group of cationic lipid contained in particle composition] / [concentration of phosphate group in nucleic acid].
- siRNA-encapsulated particle compositions 7 to 10 For the particle compositions 7 to 10, 279 ⁇ L of the particle composition was added to 100 ⁇ L of a 100 ⁇ M siRNA aqueous solution, mixed, and allowed to stand at 4 ° C. for 2 hours, whereby the siRNA was encapsulated in the particle composition. .
- the charge ratio (+/ ⁇ ) of the siRNA-encapsulated particle composition thus obtained is 8.
- the siRNA-encapsulated particle compositions obtained from the particle compositions 7 to 10 were used in various tests described later by preparing a stock that had been lyophilized according to a conventional method and redissolving it in water.
- siRNAs used in the following test examples can be obtained through Japan Easy Tea Co., Ltd.
- SiRNA (Luc) designed to target the Cypridina luciferase gene, using 5′-CUUACGCUGAGUACUCUCGAdTdT-3 ′ (SEQ ID NO: 1) as the sense strand and 5′-UCGAAGUAUCUCAGCGUAAGdTdT-3 ′ (SEQ ID NO: 2) as the antisense strand,
- This siRNA has a double chain formed by a conventional method.
- SiRNA (Plk1) Designed to target human Plk1 (Polo-like kinase 1) gene, 5′-CCAUAUAACGAGCUUGCUUAAdTdT-3 ′ (SEQ ID NO: 3) as sense strand and 5′-UUAAGCAGCUGUAAUGGdTdT-3 ′ as antisense strand SiRNA in which a double chain is formed by a conventional method using SEQ ID NO: 4).
- the Plk1 gene is an important kinase in the M phase of cell division.
- siRNA (Plk1) induces apoptosis when introduced into cells.
- F-siRNA (Luc) formed in siRNA (Luc) using an antisense strand (5′-Cy3-UCGAAGUACUCAGCGUAAGdTdT-3 ′) with a Cy3 label attached to the 5 ′ end of the antisense strand of SEQ ID NO: 2.
- Test Example 5a the particle composition 4 and the particle composition 6 are used as the particle composition, and siRNA (Luc) is used as the siRNA so that the charge ratios are 0.5, 1, 2, 4, and 8.
- SiRNA inclusion treatment was performed by the method shown in Experimental Example 5. The inclusion rate of siRNA in each particle composition after the encapsulation treatment was analyzed by electrophoresis as follows. Each particle composition containing 100 ng of siRNA was loaded onto a polyacrylamide gel (Novex 20% TBE Gel, Invitrogen), and electrophoresis was performed using a TBE solution as an electrophoresis buffer under an applied voltage of 100 V and an electrophoresis time of 1 hour. went. As a control, 100 ng of siRNA was run simultaneously. After completion, staining was performed with a coloring reagent SYBR (registered trademark) Green II (Invitrogen), and imaged with an imaging device for image analysis Molecular Imager FX (Bio-Rad).
- SYBR registered trademark
- Green II Invitrogen
- FIG. 4 is a diagram showing the results of electrophoresis in Test Example 5a.
- a charge ratio of 0 means a control.
- a free siRNA-derived band was not confirmed. This means that when the particle composition 4 and the particle composition 6 were used, almost all siRNA could be included in the particle composition when the charge ratio was 2 or more. Since the particle composition containing siRNA has a lower electrophoretic mobility than siRNA, when siRNA is appropriately included, a band derived from free siRNA cannot be detected in the corresponding electrophoresis lane.
- Test Example 5b Particle Compositions 3 to 10 and Comparative Particle Composition C3 were used as the particle composition, and siRNA (Luc) was used as the siRNA.
- SiRNA inclusion treatment was performed by the method. 10 mM HEPES buffer (pH 7.4) was added to the particle composition after the encapsulation treatment, and the concentration of the charged lipid was adjusted to 0.1 mg / mL. About these samples (800 microliters), zeta potential was measured using light scattering particle size measuring device Zetasizer Nano ZS (Malvern Instruments). A disposable capillary cell (DTS1060, Malvern Instruments) was used for the measurement, and the temperature at the time of measurement was set to 25 ° C.
- DTS1060 Malvern Instruments
- the encapsulated particle composition obtained using the particle compositions 3 to 10 has a significantly reduced zeta potential absolute value, and many siRNAs are encapsulated.
- the ratio of charged lipid added to the block copolymer unit increased, the reduction range of the zeta potential after siRNA inclusion treatment was large. This means that the larger the added ratio of charged lipid, the more siRNA can be encapsulated.
- Test Example 5c Activity evaluation against MDA-MB-231 cells
- the particle compositions 3 to 6 were used as the particle composition
- siRNA (Plk1) was used as the siRNA
- siRNA inclusion treatment was performed by the method shown in Experimental Example 5 so that the charge ratio was 8. went.
- the siRNA-encapsulated particle composition the following activity evaluation on MDA-MB-231 cells was performed. Similar experiments were performed using siRNA (Luc) as an inactive control sequence.
- siRNA-encapsulated particle composition 10 mM HEPES buffer (pH 7.4) was added to the siRNA-encapsulated particle composition, and the siRNA concentration was adjusted to 3 ⁇ M / mL.
- Human breast cancer-derived MDA-MB-231 cells were seeded in a 96-well dish at a rate of 2000 cells per well, and 24 hours later, each siRNA-encapsulated particle composition was added to the medium.
- the final concentration of siRNA in the medium was adjusted to 300 nM, 100 nM, 33 nM and 11 nM. After further culturing for 96 hours, the cell viability was evaluated using Cell Counting Kit-8 (Dojindo).
- FIG. 5 is a graph for explaining the results of this activity evaluation.
- the particle composition containing siRNA (Plk1) had a remarkable decrease in the number of cells compared to the particle composition containing siRNA (Luc). This means that the siRNA-encapsulated particle composition functioned normally.
- Test Example 5d Evaluation of aggregation in serum
- the particle compositions 3 to 10 and the comparative particle composition C3 were used as the particle composition
- siRNA (Luc) was used as the siRNA, so that the charge ratio was 8 and shown in Experimental Example 5.
- SiRNA encapsulation was performed by the method described above. From the measurement samples A and B prepared as described above, the absorbance with respect to light having a wavelength of 700 nm was measured with a plate reader (POWERSCAN HT, Dainippon Pharmaceutical), and the degree of blood aggregation was calculated. In addition, when the obtained value was 0 or less, the blood aggregation degree was set to 0.
- FIG. 6 shows measurement sample A (with FBS) and measurement sample B (without FBS) in the encapsulated particle composition using particle composition 4 and the encapsulated particle composition using comparative particle composition C3. It is a graph which shows the obtained light absorbency. As shown in FIG. 6, in the encapsulated particle composition using the comparative particle composition C3, the absorbance was greatly increased by the FBS treatment. This means that aggregation has occurred due to interaction with serum components. In the encapsulated particle composition (siRNA-encapsulated particle composition) using the particle composition 4, there was almost no increase in absorbance due to the FBS treatment.
- FIG. 7 and Table 8 below are graphs showing the relationship between the absolute value of the zeta potential of the encapsulated particle composition measured in Test Example 5b and the blood aggregation level of the encapsulated particle composition measured in Test Example 5d. And a table.
- the encapsulated particle composition has a remarkable degree of blood aggregation when the absolute value of the zeta potential is in the range of 5 mV or less, 3 mV or less, further 2 mV or less, particularly 1 mV or less. It was low.
- Test Example 5e Evaluation of blood retention
- the particle compositions 3 to 10 were used as the particle composition, and F-siRNA (Luc) was used as the siRNA, so that the charge ratio was 8, and the siRNA was synthesized by the method shown in Experimental Example 5.
- the inclusion process was performed.
- siRNA inclusion treatment was performed so that the charge ratio was 1, 2, and 4.
- a 3M sodium chloride aqueous solution was added to the obtained siRNA-encapsulated particle composition at a volume ratio of 1/20 to prepare an isotonic solution (siRNA-encapsulated particle composition sample) containing 150 mM sodium chloride.
- siRNA simple substance sample As a control, 3M sodium chloride aqueous solution was added at a volume ratio of 1/20 to a 10 ⁇ M siRNA solution obtained by dissolving F-siRNA (Luc) in 10 mM HEPES buffer at pH 7.4, and 150 mM sodium chloride was added.
- the containing isotonic solution (siRNA simple substance sample) was prepared.
- the siRNA-encapsulated particle composition sample and the siRNA simple substance sample were administered to the tail vein of a Balb / c mouse (Nippon Charles River), and 200 ⁇ L of blood was collected from the inferior vena cava 1 hour later. The dose of each sample was adjusted so that the ratio of F-siRNA to mouse body weight was 1 mg / kg.
- the blood collected from individuals administered with samples derived from the particle compositions 3 to 6 was centrifuged at 4 ° C. and 2000 ⁇ g for 10 minutes to obtain 80 ⁇ L of plasma from the supernatant.
- the plasma fluorescence intensity was measured with a plate reader (POWERSCANSHT, Dainippon Pharmaceutical Co., Ltd.) (excitation wavelength: 485 nm, fluorescence wavelength: 528 nm) to quantify F-siRNA remaining in the blood.
- Blood collected from individuals administered with samples derived from the particle compositions 7 to 10 was centrifuged at 4 ° C. and 2000 ⁇ g for 10 minutes to obtain 100 ⁇ L of plasma from the supernatant.
- the siRNA-encapsulated particle composition prepared using the particle compositions 3 to 10, 0.9% or more of F-siRNA remained in the blood.
- the higher the mass ratio of the block copolymer unit to the charged lipid the higher the residual rate of F-siRNA. This is because when the charged lipid content in the particle composition is constant, the period in which the drug can be maintained in the particle in the blood increases as the density of the region constituted by the hydrophilic polymer chain segment increases. It means to become.
- the sample with a lower absolute value of the zeta potential has a higher residual amount of F-siRNA, and the residual amount of F-siRNA in the sample using the particle composition 7 having an absolute value of the zeta potential of 0.37 mV is 37.9. %, And the amount of F-siRNA remaining in the sample using the particle composition 9 having an absolute value of the zeta potential of 0.57 mV was 15.5%.
- Table 10 the higher the charge ratio in the siRNA-encapsulated particle composition, the higher the residual rate of F-siRNA. This means that the larger the added ratio of charged lipid, the longer the period during which the drug can be maintained in the particles in the blood.
- the sample using the particle composition 7 was remarkably superior in the retention of the encapsulated drug in the blood, although the absolute value of the zeta potential was comparable to the sample using the particle composition 6. .
- the particle composition by constituting the particle composition with an anionic polymer, it is possible to greatly improve the retention of the encapsulated drug in the blood compared to the case where the particle composition is constituted with a neutral polymer.
- Test Example 5f Evaluation of organ transferability
- the particle composition 3 and the comparative particle composition C3 were used as the particle composition
- F-siRNA (Luc) was used as the siRNA, so that the charge ratio was 8 and shown in Experimental Example 5 SiRNA encapsulation was performed by the method described above.
- An isotonic solution (encapsulated particle composition sample) containing 150 mM sodium chloride was prepared by adding a 3M sodium chloride aqueous solution at a volume ratio of 1/20 to the particle composition after the encapsulation treatment.
- Balb / c nude mice female, 5 weeks old, Charles River, Japan
- the encapsulated particle composition sample was administered to mice whose tumor size was 200 mm 3 or more after 4 weeks.
- the amount of F-siRNA administered was adjusted to 1 mg / kg relative to the body weight of the mouse. 50 to 100 ⁇ g of each organ was collected 10 minutes, 60 minutes and 180 minutes after administration, and 1 mL of RNA extraction reagent Sepazol RNA I (Nacalai Tesque) was added and homogenized. After adding 100 ⁇ L of chloroform to 500 ⁇ L of each homogenate and centrifuging for 10 minutes at 5 ° C.
- the fluorescence intensity of 200 ⁇ L of the supernatant was measured with a plate reader (POWERSCAN HT, Dainippon Pharmaceutical) (excitation wavelength 485 nm, fluorescence wavelength 528 nm). F-siRNA transferred into each organ was quantified.
- FIG. 8 is a graph for explaining organ transferability of the drug investigated in Test Example 5f.
- the encapsulated particle composition obtained using the comparative particle composition C3 it disappeared quickly from the blood and significant accumulation in the lung was observed.
- the siRNA-encapsulated particle composition obtained using the particle composition 4 it stayed in the blood for a longer time and was also transferred to the tumor.
- the particle composition of the present invention as a drug carrier and encapsulating the drug to obtain a pharmaceutical composition
- the drug especially a polymer compound
- the drug can be more effectively retained in the living body and protected from degradation and excretion. it can.
- the drug can be delivered to the affected area while maintaining the activity.
- aggregation of the drug carrier due to interaction with blood components can be avoided.
- the reduction of the dosage is useful also from the viewpoint of inducing an immune response to proteins, nucleic acids and the like and suppressing side effects such as effects on the blood coagulation system.
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Abstract
Description
i)pHが7.4である10mMのHEPESバッファー溶液に、荷電脂質3の総量が当該バッファー溶液1mLあたり2.2mgとなるように測定対象の組成物(粒子組成物1または医薬組成物1’)を添加し、更にFBS(ウシ血清)を当該バッファー溶液1mLあたり9mLとなるように添加して、測定サンプルAを調製する。
ii)FBS(ウシ血清)に代えてpHが7.4である10mMのHEPESバッファー溶液を用いること以外は、上記i)と同様にして、測定サンプルBを調製する。
iii)測定サンプルAおよびBを、37℃で24時間静置した後、波長700nmの光線に対する吸光度をそれぞれから測定する。
iv)「測定サンプルAより得た吸光度」から「測定サンプルBより得た吸光度」を減算して得られる値を、血中凝集度とする。なお、当該値が小さいほど血中において測定対象組成物が凝集しにくい。
[実施例1-1]
質量平均分子量(Mw)10000のα-メトキシ-ω-アミノ-ポリエチレングリコール(以降、「PEG」と表示する場合がある)(日油株式会社)5gをジメチルスルホキシド50mLに溶解し、γ-ベンジル-L-グルタメート(以降、「PBLG」と表示する場合がある)のN-カルボン酸無水物(NCA)5.5g(ポリエチレングリコールに対し42等量)を加え、40℃で24時間反応を行った。この反応溶液をヘキサン及び酢酸エチルの混合溶媒(体積比1:1)1L中へ滴下することで、ポリマーを析出させた。減圧濾過によりポリマーを回収し、さらに乾燥させることで、8.6gの固形物を得た。これをDMF86mLに溶解し、無水酢酸432μLを加え、40℃で24時間反応を行った。反応溶液をヘキサン及び酢酸エチルの混合溶媒(体積比1:1)1L中へ滴下することで、ポリマーを析出させた。ポリマーは減圧濾過により回収し、さらに乾燥させることで、8.1gのポリエチレングリコール-ポリ(γ-ベンジル-L-グルタメート)-Acブロックコポリマー(以降、「PEG-PBLG」と表示する場合がある)を得た。PEG-PBLGの構造式を下記に示す。1H-NMRによる解析から、PBLGブロックの重合度は40であった。
DOTAPに代えてアニオン性の荷電脂質であるホスファチジン酸(以降、「PA」と表示する場合がある)を用いたこと以外は、実施例1-1と同様にして粒子組成物2を得た。
実施例1-1で得たPEG-PBLGのクロロホルム溶液(50mg/mL)4mLと、カチオン性の荷電脂質であるDOTAP(Avanti Polar Lipid)のクロロホルム溶液(40mg/mL)0.5mL及び中性の脂質であるジオレオイルホスファチジルエタノールアミン(以降、「DOPE」と表示する場合がある)(Avanti Polar Lipid)のクロロホルム溶液(40mg/mL)0.5mLを混合した後、ロータリーエバポレーターで溶媒を留去し、さらに一晩減圧乾燥した。得られた固形物に20mMのHEPESバッファー(pH7.4)5mLを加え、3時間室温で攪拌して懸濁させた。この懸濁液を超音波処理(130W、1秒パルス、20分間)することで、粒子化を行った。この溶液を0.2μmのフィルター(マイレクスGP、ミリポア)でろ過し、粒子組成物3を得た。
PEG-PBLGのクロロホルム溶液の量を2mLに変更したこと以外は、実施例1-3と同様にして粒子組成物4を得た。
PEG-PBLGのクロロホルム溶液の量を1.33mLに変更したこと以外は、実施例1-3と同様にして粒子組成物5を得た。
PEG-PBLGのクロロホルム溶液の量を1mLに変更したこと以外は、実施例1-3と同様にして粒子組成物6を得た。
実施例1-1で得られたPEG-PBLGをアルカリ処理することで、グルタミン酸側鎖のベンジル基を脱保護し、ポリエチレングリコール-ポリ(L-グルタミン酸)ブロックコポリマー(PEG-pGlu)を得た。このPEG-pGluのグルタミン酸側鎖に対し、オクチルアルコールを用いた縮合反応により部分的にオクチル基(C8H17)を導入することで、アニオン性ポリマーであるPEG-pGlu(C8)ポリマーを得た。1H-NMRによる解析から、オクチル基の導入数はポリマー当たり33個であった。PEG-pGlu(C8)の構造式を下記に示す。
PEG-pGlu(C8)のメタノール溶液(50mg/mL)の量を0.5mLに変更したこと以外は実施例1-7と同様にして、粒子組成物8を得た。
実施例1-1で得られたPEG-PBLGをアルカリ処理することで、グルタミン酸側鎖のベンジル基を脱保護し、ポリエチレングリコール-ポリ(L-グルタミン酸)ブロックコポリマー(PEG-pGlu)を得た。このPEG-pGluのグルタミン酸側鎖に対し、ベンジルアルコールを用いた縮合反応により部分的にベンジル基(PhCH2)を導入することで、アニオン性ポリマーである、PEG-pGlu(Bn)ポリマーを得た。1H-NMRによる解析から、ベンジル基の導入数はポリマー当たり34個であった。PEG-pGlu(Bn)の構造式を下記に示す。
PEG-pGlu(C8)のメタノール溶液(50mg/mL)の量を0.5mLに変更したこと以外は実施例1-7と同様にして、粒子組成物10を得た。
DOTAPを添加しないこと以外は、実施例1-1と同様にして比較用粒子組成物C1を得た。
DOTAPを添加しないこと以外は、実施例1-3と同様にして比較用粒子組成物C2を得た。
PEG-PBLGを添加しないこと以外は、実施例1-3と同様にして比較用粒子組成物C3を得た。当該組成物C3は、ブロックコポリマーを含有せず、荷電脂質であるDOTAPと中性脂質であるDOPEのみを含有する。
動的光散乱法により粒子組成物1~10及び比較用粒子組成物C1~C3の平均粒径を測定した。結果を表1に示す。
粒子組成物3~10及び比較用粒子組成物C3に10mMのHEPESバッファー(pH7.4)を加え、荷電脂質の濃度が0.1mg/mLとなるようにサンプルを調製した。光散乱粒子径測定装置Zetasizer Nano ZS(Malvern Instruments)を用い、各サンプル(800μL)についてゼータ電位を測定した。測定にはディスポーサブルキャピラリセル(DTS1060、Malvern Instruments)を用い、測定時の温度は25℃に設定した。
粒子組成物として粒子組成物3~10及び比較用粒子組成物C3を用い、上述のようにして調製した測定サンプルAおよびBから、波長700nmの光線に対する吸光度をプレートリーダー(POWERSCAN HT、大日本製薬)により測定し、血中凝集度を算出した。なお、得られた値が0以下であった場合、血中凝集度は0とした。この結果および試験例1bで測定したゼータ電位の絶対値を表3に示すと共に、図2にゼータ電位の絶対値と血中凝集度との関係を示す。
[実施例2]
ウシ由来アルブミンがFITC(フルオレセイン5-イソチオシアネート)標識されたアルブミン-FITC(Sigma Aldrich)を、20mMのHEPESバッファー(pH7.4)に溶解し、10mg/mLの溶液を形成した。このアルブミン溶液0.2mLと、粒子組成物1(40mg/mLのPEG-PBLGと4mg/mLのDOTAPとを含有する)0.5mLと、20mMのHEPESバッファー(pH7.4)0.5mLとを混合し、4℃で1晩静置することにより、アルブミン内包粒子組成物を形成した。動的光散乱法で求めた当該粒子組成物の平均粒径は98.7nmであった。なお、アルブミンは、等電点(PI)が約4.8であり、pH7.4ではアニオン性を示す生体高分子である。
粒子組成物1に代えて粒子組成物2(40mg/mLのPEG-PBLGと4mg/mLのPAとを含有する)を用いたこと以外は、実施例2と同様にしてアルブミン内包粒子組成物を形成した。動的光散乱法で求めた当該粒子組成物の平均粒径は91.6nmであった。
粒子組成物1に代えて比較用粒子組成物C1(40mg/mLのPEG-PBLGを含有する)を用いたこと以外は、実施例2と同様にしてアルブミン内包粒子組成物を形成した。動的光散乱法で求めた当該粒子組成物の平均粒径は118nmであった。
粒子組成物1に代えて比較用粒子組成物C2(40mg/mLのPEG-PBLGと2mg/mLのDOPEとを含有する)を用いたこと以外は、実施例2と同様にしてアルブミン内包粒子組成物を形成した。動的光散乱法で求めた当該粒子組成物の平均粒径は119nmであった。
実施例2、比較例2-1~比較例2-3で得たアルブミン内包粒子組成物の溶液各40μLを、10mMのHEPESバッファー(pH7.4)360μLと混合した状態で、超遠心機(Optima MAX Ultracentrifuge、Beckman Coulter)により100000×g、4℃で1時間超遠心処理した。上澄み液におけるアルブミン-FITCの蛍光強度を、プレートリーダー(POWERSCAN HT、大日本製薬)により測定(励起波長485nm、蛍光波長528nm)し、下記式(1)に基づき、粒子組成物におけるアルブミンの保持率を算出した。
A:粒子組成物に添加したアルブミン-FITCの蛍光強度
B:上澄み液におけるアルブミン-FITCの蛍光強度
実施例2及び比較例2-1で得たアルブミン内包粒子組成物の溶液各1mLを、Sepharose CL-4Bを用いたゲルろ過クロマトグラフィーにより分析した。溶出液をフラクションに分けて回収し、各フラクションに含まれるアルブミン-FITCの蛍光強度をプレートリーダー(POWERSCAN HT、大日本製薬)により測定した(励起波長485nm、蛍光波長528nm)。なお、溶離液としては、10mMのHEPESバッファーに150mMの塩化ナトリウムを添加した溶液(pH7.4)を用いた。
[実施例3]
平均分子量20000のデキストランをFITCで標識したデキストラン-FITC(Sigma Aldrich)を20mMのHEPESバッファー(pH7.4)に溶解し、10mg/mLの溶液を形成した。このデキストラン溶液0.2mLと、粒子組成物1(40mg/mLのPEG-PBLGと4mg/mLのDOTAPとを含有する)0.5mLと、20mMのHEPESバッファー(pH7.4)0.5mLとを混合し、4℃で1晩静置することにより、デキストラン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は77.6nmであった。なお、デキストランはpH7.4においてアニオン性を示す生体高分子である。
粒子組成物1に代えて粒子組成物2(40mg/mLのPEG-PBLGと4mg/mLのPAとを含有する)を用いたこと以外は、実施例3と同様にしてデキストラン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は95.4nmであった。
粒子組成物1に代えて比較用粒子組成物C1(40mg/mLのPEG-PBLGを含有する)を用いたこと以外は、実施例3と同様にしてデキストラン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は119nmであった。
粒子組成物1に代えて比較用粒子組成物C2(40mg/mLのPEG-PBLGと4mg/mLのDOPEとを含有する)を用いたこと以外は、実施例3と同様にしてデキストラン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は119nmであった。
実施例3、比較例3-1~比較例3-3で得たデキストラン内包粒子組成物の溶液各40μLにつき、試験例2aと同様にして上澄み液におけるデキストラン-FITCの蛍光強度を測定し、下記式(2)に基づき、粒子組成物におけるアルブミンの保持率を算出した。
A’:粒子組成物に添加したデキストラン-FITCの蛍光強度
B’:上澄み液におけるデキストラン-FITCの蛍光強度
[実施例4]
アルブミン(ウシ由来、Sigma Aldrich)を50mMのグリシンバッファー(pH3)に溶解し、1mg/mLの溶液を形成した。このアルブミン溶液2mLと、粒子組成物2(40mg/mLのPEG-PBLGと4mg/mLのPAとを含有する)0.5mLと、20mMのHEPESバッファー(pH7.4)0.5mLとを混合し、pH3.3、4℃で1晩静置することにより、アルブミン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は78.8nmであった。なお、アルブミンは、pH3.3ではカチオン性を示す。
粒子組成物2に代えて粒子組成物1(40mg/mLのPEG-PBLGと4mg/mLのDOTAPとを含有する)を用いたこと以外は、実施例4と同様にしてアルブミン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は95.1nmであった。
粒子組成物2に代えて比較用粒子組成物C1(40mg/mLのPEG-PBLGを含有する)を用いたこと以外は、実施例4と同様にしてアルブミン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は115nmであった。
粒子組成物2に代えて比較用粒子組成物C2(40mg/mLのPEG-PBLGと1mg/mLのDOPEとを含有する)を用いたこと以外は、実施例4と同様にしてアルブミン内包粒子組成物を形成した。動的光散乱法により求めた当該粒子組成物の平均粒径は120nmであった。
実施例4、比較例4-1~比較例4-3で得たアルブミン内包粒子組成物の溶液各400μLを、超遠心機(Optima MAX Ultracentrifuge、Beckman Coulter)により100000×g、4℃で1時間超遠心処理した。上澄み液におけるアルブミンの濃度を、タンパク質定量キットBCA Protein Assay(Pierce)により定量し、下記式(3)に基づき、粒子組成物におけるアルブミンの保持率を算出した。
A”:粒子組成物に添加したアルブミンの濃度
B”:上澄み液におけるアルブミン濃度
[実験例5]
次に記載する手順に従って各粒子組成物へのsiRNAの内包処理を行った。
・siRNA(Luc):ウミホタルルシフェラーゼ遺伝子を標的として設計され、センス鎖として5’-CUUACGCUGAGUACUUCGAdTdT-3’(配列番号1)、アンチセンス鎖として5’-UCGAAGUACUCAGCGUAAGdTdT-3’(配列番号2)を用い、常法により2重鎖を形成させたsiRNAである。
・siRNA(Plk1):ヒトPlk1(Polo-like kinase 1)遺伝子を標的として設計され、センス鎖として5’-CCAUUAACGAGCUGCUUAAdTdT-3’(配列番号3)、アンチセンス鎖として5’-UUAAGCAGCUCGUUAAUGGdTdT-3’(配列番号4)を用い、常法により2重鎖を形成させたsiRNAである。Plk1遺伝子は、細胞分裂のM期において重要なキナーゼである。siRNA(Plk1)は、細胞内に導入された場合にアポトーシスを誘導する。
・F-siRNA(Luc):siRNA(Luc)において、配列番号2のアンチセンス鎖の5’末端にCy3標識を付したアンチセンス鎖(5’-Cy3-UCGAAGUACUCAGCGUAAGdTdT-3’)を用いて形成したsiRNAである。
試験例5aでは、粒子組成物として粒子組成物4および粒子組成物6を、そしてsiRNAとしてsiRNA(Luc)を使用し、電荷比が0.5、1、2、4および8になるように、実験例5に示した方法でsiRNAの内包処理を行った。内包処理後の各粒子組成物におけるsiRNAの内包率を、次のようにして電気泳動法により分析した。ポリアクリルアミドゲル(Novex 20% TBE Gel、Invitrogen)に、100ngのsiRNAを含有する各粒子組成物をロードし、TBE溶液を泳動バッファーとして用い、引加電圧100V、泳動時間1時間の条件で泳動を行った。また、対照として、100ngのsiRNAを同時に泳動した。終了後、発色用試薬SYBR(登録商標)Green II(Invitrogen)により染色を行い、画像解析用イメージング装置Molecular Imager FX(Bio-Rad)により画像化した。
試験例5bでは、粒子組成物として粒子組成物3~10及び比較用粒子組成物C3を、そしてsiRNAとしてsiRNA(Luc)を使用し、電荷比が8になるように、実験例5に示した方法でsiRNAの内包処理を行った。内包処理後の粒子組成物に10mMのHEPESバッファー(pH7.4)を加え、荷電脂質の濃度を0.1mg/mLに調整した。これらのサンプル(800μL)について、光散乱粒子径測定装置Zetasizer Nano ZS(Malvern Instruments)を用い、ゼータ電位を測定した。測定にはディスポーサブルキャピラリセル(DTS1060、Malvern Instruments)を用い、測定時の温度は25℃に設定した。
試験例5cでは、粒子組成物として粒子組成物3~6を用い、siRNAとしてsiRNA(Plk1)を使用し、電荷比が8になるように、実験例5に示した方法でsiRNAの内包処理を行った。そして、siRNA内包粒子組成物を用いて、MDA-MB-231細胞に対する次のような活性評価を行った。なお、不活性なコントロール配列としてsiRNA(Luc)を用い、同様の実験を行った。
siRNA内包粒子組成物に10mMのHEPESバッファー(pH7.4)を加え、siRNA濃度を3μM/mLに調整した。ヒト乳癌由来のMDA-MB-231細胞を、1ウェル当たり細胞2000個の割合で96ウェルのディッシュに播き、24時間後に各siRNA内包粒子組成物を培地に添加した。siRNAの培地中での最終濃度は300nM、100nM、33nM及び11nMとなるように調整した。96時間さらに培養した後、細胞の生存率を細胞数測定キットCell Counting Kit-8(同仁堂)を用いて評価した。
試験例5dでは、粒子組成物として粒子組成物3~10及び比較用粒子組成物C3を、そしてsiRNAとしてsiRNA(Luc)を使用して、電荷比が8になるように、実験例5に示した方法でsiRNAの内包処理を行った。上述のようにして調製した測定サンプルAおよびBから、波長700nmの光線に対する吸光度をプレートリーダー(POWERSCAN HT、大日本製薬)により測定し、血中凝集度を算出した。なお、得られた値が0以下であった場合、血中凝集度は0とした。
試験例5eでは、粒子組成物として粒子組成物3~10を、そしてsiRNAとしてF-siRNA(Luc)を使用して、電荷比が8になるように、実験例5に示した方法でsiRNAの内包処理を行った。さらに、粒子組成物4については、電荷比が1、2および4になるようなsiRNAの内包処理も行った。得られたsiRNA内包粒子組成物に対し、3Mの塩化ナトリウム水溶液を体積比1/20で添加し、150mMの塩化ナトリウムを含有する等張液(siRNA内包粒子組成物サンプル)を調製した。対照として、F-siRNA(Luc)をpH7.4の10mMのHEPESバッファーに溶解して得た10μMのsiRNA溶液に、3Mの塩化ナトリウム水溶液を体積比1/20で添加し、150mMの塩化ナトリウムを含有する等張液(siRNA単体サンプル)を調製した。Balb/cマウス(日本チャールズリバー)の尾静脈にsiRNA内包粒子組成物サンプル及びsiRNA単体サンプルを投与し、その1時間後に下大静脈から血液200μLを採取した。各サンプルの投与量は、マウス体重に対するF-siRNAの比率が1mg/kgとなるように調整した。
試験例5fでは、粒子組成物として粒子組成物3及び比較用粒子組成物C3を、そしてsiRNAとしてF-siRNA(Luc)を使用して、電荷比が8になるように、実験例5に示した方法でsiRNAの内包処理を行った。内包処理後の粒子組成物に対し、3Mの塩化ナトリウム水溶液を体積比1/20で添加し、150mMの塩化ナトリウムを含有する等張液(内包処理粒子組成物サンプル)を調製した。Balb/cヌードマウス(雌、5週齢、日本チャールズリバー)にヒト乳癌由来のMDA-MB-231細胞を移植した。4週間後に腫瘍サイズが200mm3以上となったマウスに対し、内包処理粒子組成物サンプルを投与した。投与したF-siRNAの量は、マウスの体重に対し1mg/kgとなるようにした。投与から10分後、60分後、及び180分後に各臓器50~100μgを採取し、それぞれRNA抽出試薬セパゾール RNA I(ナカライテスク)1mLを加え、ホモジナイズした。各ホモジネート500μLに対しクロロホルム100μLを加え、4℃、5200Gで10分遠心した後、上澄み200μLの蛍光強度を、プレートリーダー(POWERSCAN HT、大日本製薬)により測定(励起波長485nm、蛍光波長528nm)し、各臓器中に移行したF-siRNAを定量した。
Claims (12)
- 疎水性ポリマー鎖セグメントと親水性ポリマー鎖セグメントとを有するブロックコポリマーユニットを含有し、複数の前記ブロックコポリマーユニットが前記疎水性ポリマー鎖セグメントを内側に向けると共に前記親水性ポリマー鎖セグメントを外側に向けた状態で放射状に配置された、粒子組成物であって、
内包されるべき薬物の電荷とは反対の電荷を帯びた荷電脂質をさらに有し、当該荷電脂質との静電結合によって前記薬物を粒子内に保持する一方、当該荷電脂質が前記疎水性ポリマー鎖セグメント側に引き寄せられた状態で配置されることによって、前記荷電脂質とは反対の電荷を帯びた帯電性物質を誘引し得る電荷を粒子外周面が帯びることが防止された、粒子組成物。 - 前記荷電脂質が粒子組成物の周方向において連続する状態で配置されておらず、粒子組成物の周方向において隣接する前記ブロックコポリマーユニット間に介在するように配置されることによって、粒子組成物の周方向において隣接する荷電脂質間の接触が前記ブロックコポリマーユニットによって分断された状態にある、請求項1に記載の粒子組成物。
- 前記親水性ポリマー鎖セグメントがポリエチレングリコール鎖であり、前記疎水性ポリマー鎖セグメントがポリアミノ酸鎖である、請求項1又は2に記載の粒子組成物。
- 前記疎水性ポリマー鎖セグメントがアニオン性ポリマー鎖セグメントである、請求項1~3のいずれか1項に記載の粒子組成物。
- 前記疎水性ポリマー鎖セグメントが、下記一般式(I)又は(II)で表される
- 前記一般式(I)及び(II)において、R5が-O-であり、R6が、ベンジル基、-(CH2)4-フェニル基、又は、未置換の若しくはアミノ基若しくはカルボニル基で置換されたC4~C16アルキル基である、請求項5に記載の粒子組成物。
- pHが7.4である10mMのHEPESバッファー溶液に、前記粒子組成物に含有される前記荷電脂質の総量が1ミリリットルの当該バッファー溶液あたり0.1mgになるように添加した場合に測定されるゼータ電位の絶対値が、15mV以下の範囲に制御された状態にあることによって、血中における凝集が防止された、請求項1~6のいずれか1項に記載の粒子組成物。
- 前記ゼータ電位の絶対値が3mV以下の範囲に制御された状態にあることによって、血中における凝集が更に防止された、請求項7に記載の粒子組成物。
- 請求項1~8のいずれか1項に記載の粒子組成物と、当該粒子組成物に内包された、前記荷電脂質とは反対の電荷を帯びた薬物と、を有する医薬組成物。
- pHが7.4である10mMのHEPESバッファー溶液に、前記粒子組成物に含有される前記荷電脂質の総量が1ミリリットルの当該バッファー溶液あたり0.1mgになるように添加した場合に測定されるゼータ電位の絶対値が、10mV以下の範囲に制御された状態にあることによって、血中における凝集が防止された、請求項9に記載の医薬組成物。
- 前記ゼータ電位の絶対値が2mV以下の範囲に制御された状態にあることによって、血中における凝集が更に防止された、請求項10に記載の医薬組成物。
- 前記疎水性ポリマー鎖セグメントがアニオン性ポリマー鎖セグメントで構成されていることによって、薬物の血中滞留性が向上した状態にある、請求項9~11のいずれか1項に記載の医薬組成物。
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EP (2) | EP3150194A1 (ja) |
JP (1) | JP4912510B2 (ja) |
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ES (1) | ES2614615T3 (ja) |
HU (1) | HUE031793T2 (ja) |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2015507605A (ja) * | 2011-11-22 | 2015-03-12 | オリジナル バイオメディカルズ カンパニー,リミテット | キレート化複合ミセルを有する薬物キャリア及びその応用 |
WO2017047364A1 (ja) * | 2015-09-16 | 2017-03-23 | 株式会社島津製作所 | ナノ粒子およびその製造方法 |
CN109134778A (zh) * | 2018-08-16 | 2019-01-04 | 西南民族大学 | 电荷翻转型聚合物胶束、载药胶束及其制备方法 |
JP2020111600A (ja) * | 2017-09-22 | 2020-07-27 | 旭化成ファーマ株式会社 | 液状医薬組成物の体内動態を予測する方法 |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2016137007A1 (ja) * | 2015-02-27 | 2016-09-01 | ナノキャリア株式会社 | 高分子処理剤 |
CN107249565A (zh) * | 2015-02-27 | 2017-10-13 | 那野伽利阿株式会社 | 聚合物微团载体组合物和聚合物微团组合物 |
Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06107565A (ja) * | 1992-08-14 | 1994-04-19 | Res Dev Corp Of Japan | 物理吸着型高分子ミセル医薬 |
WO1998058630A1 (en) | 1997-06-23 | 1998-12-30 | Sequus Pharmaceuticals, Inc. | Liposome-entrapped polynucleotide composition and method |
WO1999033489A1 (fr) * | 1997-12-26 | 1999-07-08 | Yamanouchi Pharmaceutical Co., Ltd. | Compositions medicinales a liberation prolongee |
JP2001504093A (ja) | 1996-10-11 | 2001-03-27 | アルザ コーポレイション | 融合性リポソーム組成物および方法 |
WO2001034115A2 (de) | 1999-11-09 | 2001-05-17 | Novosom Ag | Verfahren zur verkapselung von proteinen oder peptiden in liposomen, mit dem verfahren hergestellte liposomen und deren verwendung |
JP2001510786A (ja) * | 1997-07-22 | 2001-08-07 | フアーマシア・アンド・アツプジヨン・アー・ベー | 脂質剤及びタンパク質を含む脂質粒子の医薬組成物の製造方法 |
JP2005519063A (ja) * | 2001-12-28 | 2005-06-30 | スプラテック ファーマ インコーポレイティド | 遺伝子の発現を向上するための多価アニオン重合体及び両親媒性ブロック共重合体を含む薬剤組成物およびその使用方法 |
WO2005092389A1 (ja) | 2004-03-10 | 2005-10-06 | Kyowa Hakko Kogyo Co., Ltd. | 複合粒子および被覆複合粒子 |
JP2006056864A (ja) * | 2004-08-17 | 2006-03-02 | Yukio Nagasaki | 静電結合型ポリマー修飾リポソーム薬物キャリアの供給とその使用 |
WO2007043486A1 (ja) * | 2005-10-05 | 2007-04-19 | Tokyo Cro, Inc. | 生体適合性ブロック共重合体、その用途および製造法 |
WO2007136134A1 (ja) * | 2006-05-23 | 2007-11-29 | Nanocarrier Co., Ltd. | 疎水性薬物内包ポリマーミセルの製造方法 |
WO2008010341A1 (fr) * | 2006-07-18 | 2008-01-24 | Nanocarrier Co., Ltd. | Polypeptide physiologiquement actif, micelle de polymère ayant une protéine enfermée dans celle-ci, et procédé d'obtention de la micelle de polymère |
JP2008504827A (ja) * | 2004-07-02 | 2008-02-21 | プロチバ バイオセラピューティクス インコーポレイティッド | 免疫賦活性siRNA分子およびその使用方法 |
WO2008047948A1 (fr) * | 2006-10-19 | 2008-04-24 | Nanocarrier Co., Ltd. | Copolymère bloc pour complexe médicamenteux et composition pharmaceutique |
WO2008143339A1 (ja) * | 2007-05-17 | 2008-11-27 | Waseda University | 両親媒性分子、それを含む分子集合体及びその用途 |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6383811B2 (en) * | 1997-12-30 | 2002-05-07 | Mirus Corporation | Polyampholytes for delivering polyions to a cell |
JP4659937B2 (ja) * | 1999-11-19 | 2011-03-30 | ナノキャリア株式会社 | コア−シェル構造のポリイオンコンプレックスミセル |
GB0009773D0 (en) * | 2000-04-19 | 2000-06-07 | Univ Cardiff | Particulate composition |
KR100776557B1 (ko) * | 2001-07-13 | 2007-11-16 | 나노캬리아 가부시키가이샤 | 약물 내포 고분자 미셀의 동결 건조용 조성물 또는 미셀의제조방법 |
US20060099265A1 (en) * | 2003-03-20 | 2006-05-11 | Kazuhisa Shimizu | Micellar preparation containing sparingly water-soluble anticancer agent and novel block copolymer |
EP1648519B1 (en) * | 2003-07-16 | 2014-10-08 | Protiva Biotherapeutics Inc. | Lipid encapsulated interfering rna |
AU2004272646B2 (en) * | 2003-09-15 | 2011-11-24 | Arbutus Biopharma Corporation | Polyethyleneglycol-modified lipid compounds and uses thereof |
US20060051405A1 (en) | 2004-07-19 | 2006-03-09 | Protiva Biotherapeutics, Inc. | Compositions for the delivery of therapeutic agents and uses thereof |
JP5061349B2 (ja) | 2005-02-10 | 2012-10-31 | 国立大学法人 東京大学 | ポリカチオン荷電性ポリマー及び核酸のキャリヤーとしての使用 |
AU2007303205A1 (en) * | 2006-10-03 | 2008-04-10 | Tekmira Pharmaceuticals Corporation | Lipid containing formulations |
JP5592897B2 (ja) * | 2008-12-26 | 2014-09-17 | サムヤン バイオファーマシューティカルズ コーポレイション | アニオン性薬物含有薬剤学的組成物及びその製造方法 |
-
2010
- 2010-08-31 DK DK10812082.5T patent/DK2474306T3/en active
- 2010-08-31 JP JP2011528907A patent/JP4912510B2/ja not_active Expired - Fee Related
- 2010-08-31 EP EP16195540.6A patent/EP3150194A1/en not_active Withdrawn
- 2010-08-31 CN CN201080038549.XA patent/CN102481255B/zh not_active Expired - Fee Related
- 2010-08-31 KR KR1020127003442A patent/KR101689787B1/ko active IP Right Grant
- 2010-08-31 PT PT108120825T patent/PT2474306T/pt unknown
- 2010-08-31 HU HUE10812082A patent/HUE031793T2/en unknown
- 2010-08-31 WO PCT/JP2010/064816 patent/WO2011025036A1/ja active Application Filing
- 2010-08-31 US US13/381,841 patent/US9415059B2/en not_active Expired - Fee Related
- 2010-08-31 PL PL10812082T patent/PL2474306T3/pl unknown
- 2010-08-31 CN CN201510125151.4A patent/CN104758254B/zh not_active Expired - Fee Related
- 2010-08-31 CA CA2768651A patent/CA2768651C/en not_active Expired - Fee Related
- 2010-08-31 AU AU2010287391A patent/AU2010287391B2/en not_active Ceased
- 2010-08-31 EP EP10812082.5A patent/EP2474306B1/en not_active Not-in-force
- 2010-08-31 ES ES10812082.5T patent/ES2614615T3/es active Active
Patent Citations (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH06107565A (ja) * | 1992-08-14 | 1994-04-19 | Res Dev Corp Of Japan | 物理吸着型高分子ミセル医薬 |
JP2001504093A (ja) | 1996-10-11 | 2001-03-27 | アルザ コーポレイション | 融合性リポソーム組成物および方法 |
WO1998058630A1 (en) | 1997-06-23 | 1998-12-30 | Sequus Pharmaceuticals, Inc. | Liposome-entrapped polynucleotide composition and method |
JP2001510786A (ja) * | 1997-07-22 | 2001-08-07 | フアーマシア・アンド・アツプジヨン・アー・ベー | 脂質剤及びタンパク質を含む脂質粒子の医薬組成物の製造方法 |
WO1999033489A1 (fr) * | 1997-12-26 | 1999-07-08 | Yamanouchi Pharmaceutical Co., Ltd. | Compositions medicinales a liberation prolongee |
WO2001034115A2 (de) | 1999-11-09 | 2001-05-17 | Novosom Ag | Verfahren zur verkapselung von proteinen oder peptiden in liposomen, mit dem verfahren hergestellte liposomen und deren verwendung |
JP2005519063A (ja) * | 2001-12-28 | 2005-06-30 | スプラテック ファーマ インコーポレイティド | 遺伝子の発現を向上するための多価アニオン重合体及び両親媒性ブロック共重合体を含む薬剤組成物およびその使用方法 |
WO2005092389A1 (ja) | 2004-03-10 | 2005-10-06 | Kyowa Hakko Kogyo Co., Ltd. | 複合粒子および被覆複合粒子 |
JP2008504827A (ja) * | 2004-07-02 | 2008-02-21 | プロチバ バイオセラピューティクス インコーポレイティッド | 免疫賦活性siRNA分子およびその使用方法 |
JP2006056864A (ja) * | 2004-08-17 | 2006-03-02 | Yukio Nagasaki | 静電結合型ポリマー修飾リポソーム薬物キャリアの供給とその使用 |
WO2007043486A1 (ja) * | 2005-10-05 | 2007-04-19 | Tokyo Cro, Inc. | 生体適合性ブロック共重合体、その用途および製造法 |
WO2007136134A1 (ja) * | 2006-05-23 | 2007-11-29 | Nanocarrier Co., Ltd. | 疎水性薬物内包ポリマーミセルの製造方法 |
WO2008010341A1 (fr) * | 2006-07-18 | 2008-01-24 | Nanocarrier Co., Ltd. | Polypeptide physiologiquement actif, micelle de polymère ayant une protéine enfermée dans celle-ci, et procédé d'obtention de la micelle de polymère |
WO2008047948A1 (fr) * | 2006-10-19 | 2008-04-24 | Nanocarrier Co., Ltd. | Copolymère bloc pour complexe médicamenteux et composition pharmaceutique |
WO2008143339A1 (ja) * | 2007-05-17 | 2008-11-27 | Waseda University | 両親媒性分子、それを含む分子集合体及びその用途 |
Non-Patent Citations (1)
Title |
---|
See also references of EP2474306A4 * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015507605A (ja) * | 2011-11-22 | 2015-03-12 | オリジナル バイオメディカルズ カンパニー,リミテット | キレート化複合ミセルを有する薬物キャリア及びその応用 |
KR20170004044A (ko) * | 2011-11-22 | 2017-01-10 | (주)오리지날 바이오메디칼스 | 킬레이트 복합 미셀을 가지는 약물 전달체 및 그 응용 |
KR101815030B1 (ko) * | 2011-11-22 | 2018-01-08 | ㈜오리지날 바이오메디칼스 | 킬레이트 복합 미셀을 가지는 약물 전달체 및 그 응용 |
KR101885677B1 (ko) * | 2011-11-22 | 2018-08-06 | (주)오리지날 바이오메디칼스 | 킬레이트 복합 미셀을 가지는 약물 전달체 및 그 응용 |
WO2017047364A1 (ja) * | 2015-09-16 | 2017-03-23 | 株式会社島津製作所 | ナノ粒子およびその製造方法 |
JPWO2017047364A1 (ja) * | 2015-09-16 | 2018-07-05 | 株式会社島津製作所 | ナノ粒子の製造方法 |
JP2020111600A (ja) * | 2017-09-22 | 2020-07-27 | 旭化成ファーマ株式会社 | 液状医薬組成物の体内動態を予測する方法 |
JP7107984B2 (ja) | 2017-09-22 | 2022-07-27 | 旭化成ファーマ株式会社 | 液状医薬組成物の体内動態を予測する方法 |
CN109134778A (zh) * | 2018-08-16 | 2019-01-04 | 西南民族大学 | 电荷翻转型聚合物胶束、载药胶束及其制备方法 |
CN109134778B (zh) * | 2018-08-16 | 2021-03-09 | 西南民族大学 | 电荷翻转型聚合物胶束、载药胶束及其制备方法 |
Also Published As
Publication number | Publication date |
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DK2474306T3 (en) | 2017-02-06 |
HUE031793T2 (en) | 2017-07-28 |
PT2474306T (pt) | 2016-12-07 |
US9415059B2 (en) | 2016-08-16 |
EP2474306A1 (en) | 2012-07-11 |
CA2768651C (en) | 2017-05-16 |
CN104758254B (zh) | 2018-10-30 |
CN102481255A (zh) | 2012-05-30 |
CA2768651A1 (en) | 2011-03-03 |
CN104758254A (zh) | 2015-07-08 |
ES2614615T3 (es) | 2017-06-01 |
PL2474306T3 (pl) | 2017-06-30 |
US20120107377A1 (en) | 2012-05-03 |
AU2010287391B2 (en) | 2016-02-25 |
EP2474306B1 (en) | 2016-11-09 |
EP3150194A1 (en) | 2017-04-05 |
AU2010287391A1 (en) | 2012-02-16 |
CN102481255B (zh) | 2015-04-22 |
JPWO2011025036A1 (ja) | 2013-01-31 |
EP2474306A4 (en) | 2013-11-27 |
JP4912510B2 (ja) | 2012-04-11 |
KR101689787B1 (ko) | 2016-12-26 |
KR20120083280A (ko) | 2012-07-25 |
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